PROJECT SUMMARY The monoamines, which include dopamine, norepinephrine, and serotonin, are evolutionarily conserved neurotransmitters that modulate the activity of excitatory and inhibitory neurons throughout the entire brain, and are thus essential for diverse aspects of physiology and behavior. Abnormalities of monoamine systems contribute to numerous brain disorders including schizophrenia, depression, and Parkinson's disease. We recently developed viral-genetic tools to determine the input, output, and input?output relationships of a given neuronal population at the scale of the entire mouse brain, and discovered contrasting input?output architectures between locus coeruleus norepinephrine neurons and midbrain dopamine neurons. Here, we apply these tools to study the organization and function of the dorsal raphe (DR) serotonin system, which provides major serotoninergic input to the forebrain to regulate diverse . functions and brain states including mood, impulsivity, anxiety, as well as hunger and thirst. Using rabies-mediated trans-synaptic tracing, we previously defined the input architecture to the entire populations of DR-serotonin and DR-GABA neurons However, our unpublished work revealed considerable heterogeneity within the DR serotonin system and suggests that it consists of parallel sub- systems that differ in input, output, and neurotransmitter phenotypes. We propose that each DR serotonin sub-system may carry out a specific subset of the diverse functions ascribed to the DR-serotonin neurons. We plan to complete our characterization of the anatomical organization of the DR serotonin sub- systems, addressing the questions of how axons of each sub-system divide up the projections of the entire DR serotonin system, and what is the input?output relationship for each DR serotonin sub-system. These will lay a foundation for all future studies of DR-serotonin neurons. We also propose to identify behavioral functions of a subset of these sub-systems by manipulating and recording serotonin neuron subtypes in anxiety- and depression-like states known to involve serotonin, as well as new behavioral paradigms. Finally, because previous studies and our own unpublished data suggest a strong link between serotonin and thirst, we will explore the circuit and cellular mechanisms by which serotonin regulates thirst-motivated behavior using quantitative and sensitive assays we have established based on a technique we developed to gain genetic access of thirst-activated neurons. The integration of anatomical, physiological, and behavioral studies on genetic-, projection-, and activity-defined neuronal populations proposed here will help dissect the complex serotonin system into specific sub-systems and advance our understanding of how serotonin modulates diverse physiological functions and behaviors.